The 2014-15 outbreak of Ebola virus (EboV) in West Africa was one of unprecedented size and duration, highlighting the need for a deeper understanding of multiple aspects of the viral lifecycle for the informed development of critically needed therapeutics. Previous studies identified 3.47, a reversible small molecule inhibitor of the prototypical Zaire 1976 Mayinga EboV that targets the receptor Neimann-Pick C1 (NPC1), causes cholesterol buildup and blocks the interaction of the surface glycoprotein (GP) to NPC1-containing late endosomal/lysosomal membranes. Although multiple steps in the entry pathway have already been elucidated, to date the precise mechanism of how 3.47 blocks EboV GP-mediated entry has yet to be determined. The goals of this research proposal are: (1) To identify and map EboVs with different sensitivities to 3.47 inhibition using virological and genetic techniques, (2) To identify the binding site of 3.47 on NPC1 using a combination of chemical biology and protein biochemistry and (3) To analyze the mechanism of 3.47 action using biochemical assays to interrogate the effect of 3.47 on the GP-mediated steps of NPC1 interaction and GP2 triggering. The completion of this proposal will provide an understanding of the mechanism of action of an inhibitor of EboV GP-mediated entry. Determining the mechanism of action of 3.47 can not only reveal insights into the molecular mechanisms of EboV GP-NPC1 interaction, but also provide clues to how the EboV GP can escape potential therapeutics that target a critical step in the entry pathway.